Project Summary/Abstract Arterial stiffening is a hallmark of the aging process. However, premature stiffening is often seen in hypertension, obesity, insulin resistance, and type 2 diabetes (T2D). Both men and women are affected, but women with T2D are at greater risk. As augmented arterial stiffness is an independent predictor of cardiovascular disease (CVD), the increased susceptibility of insulin-resistant (IR) and obese women to arterial stiffening may explain their higher risk for CVD. In healthy women, estrogen signaling via estrogen receptor alpha (ER?) prevents stiffening, but these effects are blunted in over-nutrition and obesity when the presence of the ER? may be deleterious. Endothelial cell (EC) epithelial sodium channel (ENaC) expression increases in obese and IR female mice. Increased EC ENaC contributes to arterial stiffening and EC dysfunction, in part by lowering nitric oxide (NO) bioavailability. Aldosterone is a major ENaC stimulus, but other steroid hormones, specifically estrogen, also promote ENaC expression/function in various tissues. ENaC activity is regulated by the serum glucocorticoid inducible-kinase 1 (SGK-1) pathway. Elevated aldosterone and decreased NO bioavailability are characteristics of obese, IR and T2D women. Based on our prior work and most recent preliminary data, our hypothesis is that estrogen action through the EC ER? upregulates EC ENaC expression and activity, via SGK-1, exacerbating endothelial and arterial stiffening in obese, IR premenopausal females. The corollary to this hypothesis is that ENaC inhibition will have a greater impact on arterial stiffness in obese, premenopausal IR women than in obese IR postmenopausal women or age-matched obese, IR men. Consequently, ENaC inhibition will have a greater impact on arterial stiffness in premenopausal obese, IR women than in obese, IR men. We will measure arterial/EC stiffness in IR and obese EC-specific ER? and ENaC KO mice; in isolated ECs; and in a cohort of obese, IR women (pre and post-menopausal) and age- matched men, to accomplish the following Aims: 1) To determine whether EC ER? regulation of EC ENaC, via SGK-1, plays an important role in the genesis of accelerated endothelial and arterial stiffening in IR female mice and 2) to determine whether treatment with the ENaC inhibitor, amiloride, improves endothelial function and arterial stiffness in obese IR subjects in a randomized placebo-controlled trial. To date, the specific role of EC ER? regulation of ENaC expression/activation, in ensuing sex-related differences in arterial stiffness in obesity and insulin resistance, remains unexplored. This proposal aims to fill that gap and show that targeting ENaC activation holds extraordinary promise in reversing endothelial dysfunction and arterial stiffness in obesity and insulin resistance, and ultimately preventing cardiovascular disease, especially in women.